The liver plays a central role in the complex process of cholesterol homeostasis. Hepatic conversion of cholesterol to bile acids is a particularly important aspect of this process that is tightly regulated by nuclear hormone receptors. In response to an increased cholesterol load, activation of the LXRs increases bile acid production. Conversely, activation of the bile acid sensor FXR by increased bile acid levels decreases bile acid production via a nuclear receptor cascade that also includes the orphan receptors SHP and LRH-1. In addition to this repression of bile acid biosynthetic enzymes, FXR regulates expression of genes involved in bile acid transport and lipoprotein metabolism. The identification of FXR as a key regulator of cholesterol metabolism raises the important question of how exogenous FXR ligands might affect cholesterol levels. We have identified guggulsterone, the active component of a natural extract that lowers LDL cholesterol in humans, as an FXR antagonist. Strong evidence that inhibition of FXR is responsible for the cholesterol lowering effect of guggulsterone is provided by the observation that mice lacking FXR are resistant to this effect. We have also identified cafestol, a coffee oil and the most potent LDL cholesterol raising agent known, as a potential FXR agonist. We hypothesize that FXR antagonists provide a novel therapeutic approach to decreasing cholesterol levels. To test this hypothesis and further explore the function of FXR we will: 1) define the specific and overlapping functions of FXR isoforms, particularly their expression patterns and functional effects on different promoters, 2) use wild type and FXR knockout mice to test the role of FXR in the effects of guggulsterone and cafestol, and 3) define the molecular and biochemical basis for the effects of guggulsterone on cholesterol metabolism in vivo. These studies will provide new insights into the molecular basis of FXR function and its potential as a therapeutic target for modulation of cholesterol metabolism.